Poster Presentation at the Biophysical Society Meeting at Kansas City, MO (Feb 22-26, 1998). Biophys. J. 74, A189 (1998). Multiphoton microscopy (MPM) provides three-dimensional resolution for optical imaging of subcellular structures and monitoring of biological functions with which to study photochemical damage mechanisms. It is possible that damage during MPM arises from two-photon absorption events, from higher order multiphoton events, or from one-photon events. Doses (Int) are defined as the nth power of the peak irradiance at the sample multiplied by the total dwell time of the focal volume in a cell. (The irradiance, I, is in photons/s((m2, and n is the number of photons required for a single excitation event, or the excitation order.) To detect the minimal observed photodamage, we have selected a proliferation assay that is sensitive to perturbations of cellular metabolism and defense mechanisms as well as to dead cells. Using this assay, we are generating an intrinsic action spectrum of photodamage by monitoring the onset of DNA synthesis inhibition in cultured HeLa cells without added dyes. Sin ce we have not yet determined the excitation order of damage at all wavelengths, this action spectrum is expressed with n = 2, defining two-photon events. The measured damage thresholds are orders of magnitude above typical imaging doses. We can evaluate this data to determine the excitation order, n, using the general dose-response equation , where S is the normalized survival and the dose, D, is defined as Int. Preliminary analysis suggests that the observed damage is arising from different absorbing species at different wavelengths -- at 700 nm damage appears to be from one-photon events and at 740 nm it appears to be from two-photon events. Supported at DRBIO by the NIH (RR04224) and NSF (BIR 8800278).